Solis software

Epicardial fNADH images were analyzed using SOLIS software (Andor Technology). Raw NADH fluorescence (rNADH) was averaged at each time point within a large region of interest on the free wall of the left ventricle. Because only hearts subjected to ischemia experienced changes in NADH, these signals were normalized from 0 to 1, where 0 was the minimum rNADH value and 1 was the maximum rNADH value for a given heart. The resulting normalized NADH (nNADH) values were calculated as nNADH = (rNADH − rNADH_min)/(rNADH_max − rNADH_min). For control flow, nNADH was calculated as percent change from the minimum rNADH, which always occurred during baseline flow, such that control nNADH = (rNADH − rNADH_min)/rNADH_min.

Fluorescent Ca²⁺ activity was recorded in wide-field on a fixed-stage upright microscope (modified Olympus BX51), illuminated by a metal halide light source (PhotoFluor II, 89North) or a blue (470 nm) LED light source (M470L2, Thorlabs). Red and green channel fluorescence was visualized using a dual-bandpass filter set (Chroma 59022: excitation dual bandpass 450–490 nm/555–590 nm, emission dual bandpass 500–543 nm/603–665 nm). Red and green channels (for Alexa Fluor 568 hydrazide; Fluo-8, AM; jGCaMP7f/s) were separated during acquisition by manually exchanging an additional excitation filter in the light path (Semrock FF01: bandpass 565–605 nm; Semrock FF02: bandpass 457–487 nm). Time series acquisition was performed with a sCMOS camera (Neo DC-152Q, Andor Technology) controlled by SOLIS software (Andor Technology). Imaging protocols employed 10× (NA 0.3), 20× (NA 0.5), and 40× (NA 0.8) water immersion objectives.

(1) A near-infrared semiconductor laser with an excitation wavelength of 785 nm (Changchun New Industries Optoelectronics Tech, China). (2) A fiber optic Raman probe (Em Vision, America): the Raman fiber optic bundle was 3 m in length and had an outer nylon protective sleeve composed of 7 collection fibers (300 μm in diameter, N.A. = 0.22) surrounding the central excitation fiber (272 μm in diameter, N.A. = 0.22), with two filters incorporated at the proximal and distal ends of the probe to maximize the collection of tissue Raman signals while reducing interference from light other than Raman-scattered light. (3) A Raman imaging spectrometer (Andor, England). (4) A charge-coupled device (CCD) camera (Andor, England). (5) A monitor (Lenovo, China). (6) Andor SOLIS software. The fiber optic Raman spectroscopy system is shown in Figure 1.

Degree of cardiopatch vascularization within dorsal window chambers was assessed on days 7 and 14 post implantation. Mice were anesthetized by nose cone inhalation of isoflurane and placed on a heating pad under a microscope objective. Hyperspectral brightfield image sequences (10 nm increments from 500 to 600 nm) were captured at ×5 magnification using a tunable filter (Cambridge Research & Instrumentation, Inc.) and a DVC camera (ThorLabs), as previously described^{43 (link),44 (link)}. A custom MATLAB (MathWorks) script was applied to create maps of total hemoglobin concentration, which were further processed using local contrast enhancement in ImageJ (CLAHE plugin, FIJI) and thresholded to binary images to identify vessel area and calculate blood vessel density (BVD, total area of blood vessels per patch area; Supplementary Fig. 17).
Spontaneous Ca²⁺ transients were recorded in real-time immediately after imaging of blood vessels while mice were still under anesthesia. Fluorescent gCaMP6 signals in implanted patches were imaged through a FITC-filter using a fast fluorescent camera (Andor, at 16 μm spatial and 20 ms temporal resolution). Amplitudes of spontaneous Ca²⁺ transients were determined using the Solis software (Andor) by averaging relative fluorescence intensity (dF/F = [F_peak− F_base]/F_base) from three ~400 × 400 μm² regions within each patch^{44 (link)}.